408 M'LAUGHLIN AND WHITMORE. 



classified by us as nonchole ra, do not develop ag glut-inability to cholera 

 serum when treated as described by Zlatogorojf. 



The vibrios isolated by him from water and which "developed" agglu- 

 tinahility with cholera serum in a dilution of 1-10,000 must have been 

 cholera vibrios. 



For instance, his culture No. 15, cited above; with a dose of J slant, on Nov- 

 ember 6 showed no agglutination; J of a slant was injected into guinea pig. 

 The guinea pig died on November 9. One transplant was made on November 10 

 and the agglutination was 1-200. 



On the other hand, we have not been able at all to increase agglu- 

 tinability. 



Preparation of sera. — Sera were prepared with 10 cholera strains, 2 

 El Tor strains, and from 9 honcholera vibrios in the following manner: 

 Eighteen-hour cultures on agar were used; suspensions were made with 

 salt solution. Each rabbit received 4 or 5 injections; the interval 

 between injections usually being seven to eight days. The amounts 

 injected were as follows : 



First day, 1 loop heated one hour at 60°C, into ear vein. 

 Eighth day. 3 loops heated one hour at 60°C, into ear vein. 

 Sixteenth day, 5 loops heated one hour at 60°C, into ear vein. 

 Twenty- fourth day, 4 slant, living, intraperitoneally. 

 Tb'rty-second to thirty-fourth day, 1 slant, living, intraperitoneally. 

 On the fortieth to forty-fourth day the animal was bled. 



Selection of an animal for the preparation of a specific serum. — ■ 

 Kolle and Gotschlich (4) tested the normal sera of rabbit, ass, horse, 

 goat and ox upon vibrios and found that normal rabbit and ass sera 

 had a very slight agglutinating action upon vibrios, while horse and 

 goat sera sometimes exert an agglutinating action in a dilution of 1-40 

 or 1-50. The small quantity of serum obtainable from a rabbit is 

 usually considered a great disadvantage, but we found that large amounts 

 of good serum could be obtained by the following procedure: 



The bleeding apparatus consisted of an ordinary, large test tube with a 

 rubber cork perforated to admit two glass tubes. A short piece of rubber tubing 

 and a' long slender needle is attached to one of these glass tubes; the other is 

 connected with the vacuum pipe by about a meter of heavy rubber tubing. After 

 sterilization, the needle is inserted below the ensiform cartilage and a trifle 

 to the left, and then pushed upward into the pericardium and heart. After 

 passing through the integument the needle in passing upward meets very 

 slight resistance until the pulsating heart is encountered, but with a slight 

 additional pressure it passes through the ventricular wall and blood begins 

 to drip into the tube. The vacuum is turned on to facilitate the flow. In this way 

 10 to 15 cubic centimeters can be obtained without trouble at one bleeding. 



Eabbits may be bled upon three successive days, the product of the 

 three bleedings being combined and tested at the same time. In this 



